Reusable solid support for oligonucleotide synthesis

ABSTRACT

A reusable linker arm for solid support oligonucleotide synthesis, the linker arm comprising formula (a) wherein Z is a linker moiety and T is an organic radical. A method for adding one or more nucleosides on the linker arm is also described

This application is a 371 application of PCT/CA99/00600, filed Jun. 30,1999 which claims the benefit of provisional application U.S.60/091,683, filed Jul. 2, 1998, now abandoned.

TECHNICAL FIELD

In one of its aspects, the present invention relates to a reusable solidsupport for oligonucleotide synthesis. In another of its aspects, thepresent invention relates to a process for production of such a reusablesolid support. In yet another of its aspects, the present inventionrelates to a process for use of such a reusable solid support.

BACKGROUND ART

The art of organic chemistry on solid supports is generally known. Auseful review article on this topic may be found in “Organic Chemistryon Solid Supports” by Früchtel et al., Angew. Chem. Int. Ed. Engl.,1996, 35, pgs. 17–42, the contents of which are hereby incorporated byreference.

As discussed in Früchtel et al., the art has developed automatedsolid-phase synthesis of polypeptides, oligonucleotides andoligosaccharaides. Of particular interest here is solid-phase synthesisof oligonucleotides. The following are useful review articles/textbookson this topic:

-   Beaucage et al., Tetrahedron, 1992, 48, 2223;-   Davis et al., Innovation and Perspectives in Solid Phase Synthesis    (Ed.; R. Epton), Intercept, Andover, 1992, pg. 63;-   Montserra et al., Tetrahedron, 1994, 50, 2617; and-   S. L. Beaucage et al., Tetrahedron, 1993, 49, 6123–6194;    the contents of each of which are hereby incorporated by reference.

In the solid-phase synthesis of oligonucleotides, it is known tosynthesize the oligonucleotide on an inorganic solid support bearing asuccinyl linker arm—see, for example, any of the following references:

-   Caruthers et al., Genetic Engineering, Plenum Press, New York    (1982), Vol. 4, pgs. 1–17;-   Letsinger et al., Genetic Engineering, Plenum Press, New York    (1985), Vol. 5, pg. 191;-   Froehler et al., Nucleic Acids Research, 14:5399–5407 (1986); and-   Matteucci et al., Journal of American Chemical Society,    103:3185–3186 (1981);    the contents of each of which are hereby incorporated by reference.

Typically, the succinyl linker arm has the following general formula:

Thus, the succinyl group links the growing oligonucleotide from itsterminal 3′ hydroxyl group by an ester bond to a primary amine on thesupport, which may be, for example, conventional controlled pore glass(CPG) or silica, by an amide bond. Once the desired oligonucleotide hasbeen synthesized, it is freed or cleaved from the succinyl linker armhydrolyzing the ester carbonyl group. The hydrolysis agent is usuallyconcentrated ammonium hydroxide. Typically, this reaction can take from1–4 hours to complete. With improvements to current solid-phaseoligonucleotide synthesizers, this cleavage step can represent 50% ormore of the total time require to synthesize the desiredoligonucleotide.

Another type of linker arm is disclosed in U.S. Pat. No. 5,112,962[Letsinger et al. (Letsinger)], the contents of which are herebyincorporated by reference. Letsinger teaches a linker arm for solidsupport synthesis of oligonucleotides and oligonucleotide derivativeshave the following formula:

Thus, Letsinger teaches an oxalyl linker arm which purportedly releasethe synthesized oligonucleotide or oligonucleotide derivate in a periodof 1–30 minutes in a manner that leaves the oligonucleotide fullyprotected. The oxalyl linker arm purportedly can be rapidly cleaved by5% ammonium hydroxide in methanol, ammonium hydroxide, wet tertiaryamine, triethylamine/alcohol, triethylamine/methanol,triethylamine/ethanol, aqueous trimethylamine and other bases.Unfortunately, the oxalyl linker arm of Letsinger suffers from itspurported advantage. Specifically, the present inventors have discoveredthat the oxalyl linker arm of Letsinger is susceptible to significantspontaneous hydrolysis (e.g. spontaneous hydrolysis of ˜10–40% permonth) which renders it difficult to use in commercial operations. Theoxalyl arm is also difficult to prepare because it requires using oxalylchloride, which is highly reactive, toxic and therefore dangerous.

Regardless of the specific nature of the linker arm, it is generallyaccepted in the art that the linker arm is not reusable after productionand cleavage of the desired oligonucleotide. Thus, conventional linkerarms may be regarded as non-recyclable. This is illustrated in FIG. 1which illustrates the conventional use of a succinyl linker arm for theproduction of an oligonucleotide. Thus, as illustrated, after cleavageof the desired oligonucleotide, the support is irreversibly linked tothe linker compound (i.e., the succinyl moiety) and cannot be reused.

The art is in need of a linker arm for solid support oligonucleotidesynthesis, which linker arm is recyclable. More specifically, the art isin need of a linker arm capable of repeated oligonucleotidesynthesis/cleavage.

In published International patent application WO 97/23496 [Pon et al.],the contents of which are hereby incorporated by reference, there isreported the first recyclable linker arm. This linker arm is based on aderivatized solid support having the following formula:

wherein: R⁸ is selected from the group consisting of a substituted orunsubstituted C₁–C₂₀ alkyl group, a substituted or unsubstituted C₅–C₃₀aryl group and a substituted or unsubstituted C₅–C₄₀ alkylaryl group; X³and X⁴ are the same or different and are selected from the groupconsisting of —O—, —S—, —S(O)₂— and —N(R¹²)—; R¹² is selected from thegroup consisting of a substituted or unsubstituted C₁–C₂₀ alkyl group asubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; and Y is selected from the groupconsisting of:

-   -   —CH₂—CH₂—; —CH₂—;    -   —CH₂—O—CH₂—; —CH₂—CH₂—CH₂—;    -   —CH═CH—; —CH═C(CH₃)—;    -   —C(CH₃)═C(CH₃)—; —CH₂—C(═CH₂)—; and    -   —CH₂—S—CH₂—.

While a linker arm based on the solid support described by Pon et al. isa significant advance in the art, there is still room for improvement.Specifically, the solid support described by Pon et al. has thefollowing disadvantages.

First, prior to attachment of the linker moiety, the solid support mustbe derivatized by a process comprising the step of reacting together thecompounds of Formulae I, II and III:

wherein R⁸, X³, X⁴ and Y are as defined above. Practically, thisinvolves two steps—i.e., reaction of the compound of Formula III withone of the compounds of Formulae I and II and subsequent reaction withthe other of compounds of Formulae I and II. Thus, the disadvantage isadditional labour required to effect a two-step derivatization of thesolid support.

Second, each step of the derivatization described in the previousparagraph has the potential of incompletely derivatizing each HX⁴-moietyon the support thereby increasing the likelihood of a heterogeneoussurface. Practically, it becomes necessary to block or cap underivatizedHX⁴-moieties so that the linker moiety does interact with them. Thus,the disadvantage is additional labour and cost required to effectderivatization of the solid support.

Third, a linker arm based on the derivatized support described by Pon etal. is not as resistant to partial cleavage during regeneration as aderivatized support having a more fully saturated moiety.

In light of these disadvantages, it would be desirable to have animproved recyclable solid state support material useful in theoligonucleotide synthesis. It would be especially desirable if the thelinker moiety could be attached to the support material with little orno derivatization required of the latter.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a novel solidsupport for oligonculeotide synthesis which obviates or mitigates atleast one of the above-mentioned disadvantages of the prior art.

It is another object of the present invention to provide a novel processfor producing the solid support.

It is an object of the present invention provide a novel linker arm forsolid support oligonucleotide synthesis which obviates or mitigates atleast one of the above-mentioned disadvantages of the prior art.

It is another object of the present invention to provide a novel processfor producing a linker arm for solid support oligonucleotide synthesis.

Accordingly, in one of its aspects, the present invention provides areusable linker arm for solid support oligonucleotide synthesis, thelinker arm comprising the following formula:Z-O-T

[SUPPORT]wherein Z is a linker moiety and T is an organic radical.

In another of its aspects, the present invention provides a reusablelinker arm for solid support oligonucleotide synthesis, the linker armcomprising the following formula:NUCLEOSIDE-Z-O-T

[SUPPORT]wherein Z is a linker moiety and T is an organic radical.

In yet another of its aspects, the present invention provides a processfor production of a reusable linker arm for oligonucleotide synthesishaving the following formula:Z-O-T

[SUPPORT]wherein Z is a linker moiety and T is an organic radical, the processcomprising the step of reacting together the compounds of Formulae I andII:Z-OH   (I)HO-T

[SUPPORT]  (II)wherein Z and T are as defined above.

In another of its aspects, the present invention provides a process forproduction of a reusable linker arm for oligonucleotide synthesis havingthe following formula:NUCLEOSIDE-Z-O-T

[SUPPORT]wherein Z is a linker moiety and T is an organic radical, the processcomprising the step of reacting together the compound of Formulae I, IIand III:HO-Z-OH  (I)HO-T

[SUPPORT]  (II)NUCLEOSIDE-OH  (III)wherein Z and T are as defined above.

In yet another of its aspects, the present invention provides a processfor producing an oligonucleotide having a desired sequence comprisingthe steps of:

(i) reacting a linker arm having the formula:NUCLEOSIDE-Z-O-T

[SUPPORT]wherein Z is a linker moiety and T is an organic radical, with at leastone oligonucleoside base until an oligonucleotide having the desiredsequence is produce;

(ii) cleaving the oligonucleotide having the desired sequence to producea free oligonucleotide have the desired sequence; and a used linker arm;and

(iii) recycling the used linker arm to Step (i).

As used throughout this specification, the term “oligonucleotide” isintended to have a broad meaning and encompasses conventionaloligonucleotides, backbone-modified oligonucleotides (e.g.phosphorothioate, phosphorodithioate and methyl-phophonate analogsuseful as oligotherapeutic agents) and oligonucleotide derivatives suchas oligonucleotide-peptide conjugates.

Throughout this specification, when reference is made to a substitutedmoiety, the nature of the subsitution is not specification restrictedand may be selected from the group consisting of a C₁–C₂₀ alkyl groups,a C₅–C₃₀ aryl group a C₅–C₄₀ alkaryl group.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will be described with reference tothe accompany drawing in which:

FIG. 1 illustrates a specific process pathway for conventionaloligonucleotide synthesis; and

FIGS. 2 and 3 illustrate specific preferred embodiments of the presentinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

Initially, to facilitate an understanding of the invention, referencewill be made to FIG. 1, which illustrates a conventional process forsolid support oligonucleotide synthesis.

Thus, the initial step of the process illustrated in FIG. 1 comprisesreacting a linking compound, such as succinic acid (while succinic acidis illustrated, succinic anhydride may also be used), with aconventional amine-terminated support. The reaction results in theformation of an amide linkage between the linking compound and thesupport to produce succinyl-support conjugate.

Next, the succinyl-support conjugate is reacted with a desired initialnucleoside to produce a linker arm. In the illustrated nucleoside, DMTis dimethyoxytrityl, B is the nucleobase and R′ is H (fordeoxyribonucleosides) or OR (for ribonucleosides) wherein R is H or aconventional blocking/protecting group. The reaction results in theformation of an ester linkage between the linking compound and thedesired initial nucleoside at the 3′ position of the latter.

The linker arm is then used in conventional oligonucleotide synthesis(e.g. in a conventional automated synthesizer) to produce anoligonucleotide of desired sequence attached to the linker arm.

The oligonucleotide is then cleaved from the linker by hydrolysis. Thisserves to cleave the ester bond thereby freeing the oligonucleotide andan amine-terminated, non-reusable linker arm.

The present inventors have surprisingly and unexpectedly discovered thata support having a hydroxy-terminated functionality may be combined witha conventional linking compound to produce linker arm which may used tosynthesize an oligonucleotide of desired sequence. A key feature of theinvetion is that the linker arm may be regenerated or recycled aftercleavage of the oligonucleotide of desired sequence. To the inventors'knowledge, this is the first discovery of a derivatized support whichmay be used repeatedly in oligonucleotide synthesis.

The reusable linker arm of the present invention has the followingformula:Z-O-T

[SUPPORT]wherein Z is a linker moiety and T is an organic radical.

Preferably, T contains at least one carbon.

Preferably, T is a C₁–C₃₀₀ organic moiety, more preferably a C₁–C₂₀₀organic moiety, most preferably a C₁–C₁₀₀ organic moiety.

As will be appreciated by those of skill in the art, T may be asaturated or unsaturated organic moiety. Further, T may contain one ormore heteroatoms. For example, T may comprise at least one heteroatomselected from N and O.

In one preferred embodiment, the organic moiety in T comprises at leastone moiety having the formula:

In another preferred embodiment, the organic moiety in T comprises atleast one moiety having the formula:—N(H)—.

In yet another preferred embodiment, the organic moiety in T comprisesat least one moiety having the formula:

In yet another preferred embodiment, the organic moiety in T comprisesat least one moiety having the formula:—C—O—C—.

In yet another preferred embodiment, the organic moiety in T comprisesat least one moiety having the formula:

Further, those of skill in the art will recognize that the organicmoiety in T may be unsubstituted or substituted. For examples, theorganic moiety of T may be substituted by at least one moiety selectedfrom the group comprising a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, aC₁–C₄₀ alkoxy croup, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxy group, aC₁–C₄₀ acrylate group and a C₅–C₄₀ alkylaryl group.

In one preferred embodiment, T has the formula:

wherein q and s are the same or different and each is an integer havinga value of 0–40 and r is an integer having a value of 1–200. In thisembodiment, it is further preferred that q and s are the same ordifferent and each is an integer having a value of 1–20 and r is aninteger having a value of 1–150.

In another preferred embodiment, T has the formula:

wherein a is 0 or 1, Q is an organic moiety, R⁸ is hydrogen or aprotecting group and b is an integer having a value of 0–40. In thisembodiment, a may be 0 and R⁸ may be hydrogen. Further, a may be 1 andR⁸ may be a protecting group. Non-limiting examples of protecting groupsmay be selected from the group comprising acetyl, chloroacetyl,methoxyacetyl, t-butyl phenoxyacetyl, phenoxyacetyl, trityl,methoxytrityl, dimethoxytrityl (DMT), dialkylphosphite,pivalyl-isobutyloxycarbonyl, t-butyldimethylsilyl, phenoxyacetal,9-phenylxanthen-9-yl (pixyl), tetrahydropyranyl,methoxytetrahydropyranyl, methoxymethyl, benzyloxymethyl,methoxyethoxymethyl, methylthiomethyl, dialkylphosphate, levulinyl,dimethylphenylsilyl, trimethylsilyl, isopropyl-dimethylsilyl,diisopropylmethylsilyl, diethylisopropylsilyl, triisopropylsilyl,acetyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl,o-nitrobenzyl, o-hydroxystyryldimethylsilyl, 2-oxo-1,2-diphenylethyl,allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl,dimethoxybenzoin, dimethoxybenzoin carbonate, methylnitropiperonylcarbonate, fluorenyl-methoxycarbonyl, 2-phenylsulfonylethoxycarbony,fluorophenyl-methoxypiperidinyl and mixtures thereof.

In this embodiment, Q may be a moiety having the formula:

wherein q, r, s, t and u are the same or different and each is aninteger having a value of 0–40 and R^(a) is selected from the groupcomprising hydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ arylgroup, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxygroup, a C₂–C₄₀ acrylate group and a C₅–C₄₀ alkylaryl group. Preferably,s is 0, q, r and u are the same or different and each is an integerhaving a value of 1–10, t is an integer of 1–5 and R^(a) is hydroxyl.

In yet another preferred embodiment, T has the formula:

wherein a is 0 or 1, Q is an organic moiety, R⁸ is selected from thegroup comprising hydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₁–C₄₀ arylgroup, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxygroup, a C₁–C₄₀ acrylate group and a C₅–C₄₀ alkylaryl group, and b is aninteger having a value of 0–40. Preferably, Q is a C₁–C₁₀₀ organicmoiety. As will be appreciated by those of skill in the art, Q may be asaturated organic moiety or an unsaturated organic moiety.

It is preferreed that Q is a C₁–C₁₀₀ organic moiety comprising at leastone heteroatom selected from N and O.

In one preferred embodiment, the organic moiety Q comprises at least onemoiety having the formula:

In another preferred embodiment, the organic moiety Q comprises at leastone moiety having the formula:N(H)—.

In yet another embodiment, the organic moiety Q comprises at least onemoiety having the formula:

In yet another embodiment, the organic moiety Q comprises at least onemoiety having the formula:—C—O—C—.

In yet another embodiment, the organic moiety comprises at least onemoiety having the formula:

As will be appreciated by those of skill in art, the organic moiety Qmay unsubstituted or substituted. For example, the organic moiety Q maybe substituted by at least one moiety selected from the group comprisinga C₁–C₄₀ alkyl group, a C₁–C₄₀ aryl group, a C₁–C₄₀ alkoxy group, aC₁–C₄₀ ester group, a C₁–C₄₀ hydroxy group, a C₁–C₄₀ acrylate group anda C₁–C₄₀ alkylaryl group.

In one preferred embodiment, Q has the formula:

wherein each of x, y and z is an integer having a value of 1–40.

In the above formula for the present linker arm, Z is a linker moiety.As will be discussed below, Z is derived from a linker compound have thegeneral formula HO-Z-OH (Formula I below). The nature of the linkercompound is not particularly restricted.

In one preferred embodiment, linker moiety Z has the formula:

As will be apparent to those of skill in the art, this linker moiety maybe derived from succinic acid or succinic anhydride.

In another preferred embodiment, linker moiety Z has the followingformula:

As will be apparent to those of skill in the art, this linker moiety maybe derived from diglycolic acid or diglycolic anhydride.

In yet another preferred embodiment, linker moiety Z has the followingformula:

As will be apparent to those of skill in the art, this linker moiety maybe derived from oxalic acid or oxalyl chloride.

In yet another, and most, preferred embodiment, linker moiety Z has thefollowing formula:

wherein: R¹, R² and R³ are the same or different and are selected fromthe group consisting of hydrogen, halide, a substituted or unsubstitutedC₁–C₂₀ alkyl group, a substituted or unsubstituted C₁–C₃₀ aryl group anda substituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁴ and R⁵ are thesame or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, asubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; X¹ is selected from the groupconsisting of —O—, —C(O)—, —S—, —S(O)₂— and —N(R)—; R is selectedhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, asubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; n is 0, 1 or 2; and one of A¹ andB¹ is selected from the group consisting of hydrogen, halide, asubstituted or unsubstituted C₁–C₂₀ alkyl group, a substituted orunsubstituted C₅–C₃₀ aryl group and a substituted or unsubstitutedC₅–C₄₀ alkylaryl group, and the other of A¹ and B¹ has the formula:

wherein p is 0 or 1, X² is selected from the group consisting of —O—,—S—, —C(O)—, —S(O)₂— and —N(R)—, R is selected from the group comprisinghydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, asubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group, R⁶ and R⁷ are the same ordifferent and are selected from the group consisting of hydrogen, asubstituted or unsubstituted C₁–C₂₀ alkyl group, a substituted orunsubstituted C₅–C₃₀ aryl group and a substituted or unsubstitutedC₅–C₄₀ alkylaryl group, and m is 0, 1 or 2. In this embodiment, B¹preferably is selected from the group consisting of hydrogen, halide, asubstituted or unsubstituted C₁–C₂₀ alkyl group, a substituted orunsubstituted C₅–C₃₀ aryl group and a substituted or unsubstitutedC₅–C₄₀ alkylaryl group. Preferably, at least one, more preferably each,of R, R⁴, R⁵, R⁶ and R⁷ is hydrogen and preferably at least, morepreferably both, of m and n are 1. It is further preferred that each ofR¹, R² and R³ is hydrogen and that X¹ and X² are both —O—. Thus, in thisembodiment, the most preferred form of linker moiety Z is derived fromhydroquinone-O,O′-diacetic acid.

In yet another preferred embodiment, linker moiety Z has the followingformula:

wherein R⁴, R⁵, R⁶ and R⁷ are the same or different and are selectedfrom the group consisting of hydrogen, a substituted or unsubstitutedC₁–C₂₀ alkyl group, a substituted or unsubstituted C₅–C₃₀ aryl group anda substituted or unsubstituted C₅–C₄₀ alkylaryl group, Y is selectedfrom the group consisting of O, S, SO₂ and O—((CH₂)₁—O)_(q), 1 is aninteger less than or equal to 60, q is an integer in the range of1–1000, n and m are the same or different and are 1 or 2, with theproviso that, when Y is O, at least one of n and m is 2. Preferably, 1is an integer in the range of 1–10, and q is an integer in the range of1–000. In this embodiment, the most preferred form of linker moiety Z isderived from thiodiglycolic acid (i.e. R⁴═R⁵═R⁶═R⁷═H, n=m=1 and Y═S).

The SUPPORT in the above formula is a conventional solid support. Thenature of the solid support is not particularly restricted and is withinthe purview of a person skilled in the art. Thus, the solid support maybe an inorganic substance. Non-limiting examples of suitable inorganicsubstances may be selected from the group consisting of silica, porousglass, aluminosilicates, borosilicates, metal oxides (e.g. aluminumoxide, iron oxide, nickel oxide) and clay containing one or more ofthese. Alternatively, the solid support may be an organic substance suchas a cross-linked polymer. Non-limiting examples of a suitablecross-linked polymer may be selected from the group consisting ofpolyamide, polyether, polystyrene and mixtures thereof. The preferredsolid support for use herein is conventional and may be selected fromcontrolled pore glass bead or polystyrene beads. Further, the supportmay be either in particle form (e.g., beads), three-dimensional slabs(e.g., polymeric inserts and foams) or in a flat two-dimensional likeformat (e.g., plastic sheets, glass chips, silicon wafers, etc.). Thematerial used for the support may also be soluble in certain solvents(e.g., liquid-phase supports), but can be precipitated or crystallizedfrom other solvents.

The reusable linker of formula:Z-O-T

[SUPPORT](again, Z is a linker moiety and T is an organic radical), may then bereacted with a conventional nucleoside-linker compound to produceanother linker arm according to the present invention. This other linkerarm has the following formula:NUCLEOSIDE-Z-O-T

[SUPPORT]wherein Z is a linker moiety and T is an organic radical. The discussionherein above with respect to Z and T applies equally here. Preferably,in the above formula, NUCLEOSIDE is a moiety selected from one of thefollowing formulae:

wherein R⁸ and R¹⁰ are the same or different and are hydrogen or aprotecting group, R⁹ is hydrogen (for deoxyribonucleosides or DNA) or—OR¹¹ (for ribonucleosides or RNA) wherein R¹¹ is hydrogen or aprotecting group, and B* a nucleic acid base. Thus, in the case of RNA,there are two hydroxyl groups which may be protected. Also, the linkercan be attached to either the 5′-, 3′- or (if ribose) 2′-hydroxylpositions. Indeed, for RNA sequences, it makes little difference whetherthe ester linker formed between the nucleoside and the linker compoundis at the 2′- or 3′-hydroxyl position of the nucleoside. Thus, those ofskill in the art will recognize that the nucleoside may be protected orblocked at the various of its hydroxyl moieties.

Non-limiting examples of useful protecting groups may be selected fromthe group consisting of acetyl, chloroacetyl, methoxyacetyl, t-butylphenoxyacetyl, phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl(DMT), dialkylphosphite, pivalyl-isobutyloxycarbonyl,t-butyldimethylsilyl, phenoxyacetal, 9-phenylxanthen-9-yl (pixyl),tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl,benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl,dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl,isopropyldimethylsilyl, diisopropylmethylsilyl, diethylisopropylsilyl,triisopropylsilyl, acetyl, benzoyl, pivaloyl, trifluoroacetyl, allyl,benzyl, o-nitrobenzyl, o-hydroxystyryldimethylsilyl,2-oxo-1,2-diphenylethyl, allyloxycarbonyl, monomethoxymethyl,nitroveratryloxycarbonyl, dimethoxybenzoin, dimethoxybenzoin carbonate,methylnitropiperonyl carbonate, fluorenyl-methoxycarbonyl,2-phenylsulfonylethoxycarbony, fluorophenyl-methoxypiperidinyl and thelike.

As is known in the art, the main prerequisite for the protecting groupused on the 5′-hydroxyl position is its ability to be selectivelyremoved without causing cleavage of the linker arm. Thus, the preferredprotecting group for desired 5′-hydroxyl position(s) is the acid labiledimethoxytrityl group. The main prerequisite for protecting groups onother hydroxyl positions, is stability to the conditions used forremoval of the above protecting group. These latter protecting groupsmay be removed by the same conditions used to cleave the linker(discussed below) or separate conditions. The preferred protectinggroups for these positions are trialkylsilyl (i.e. t-butyldimethylsilyl)or acetyl. Additional information may be obtained from the followingreferences:

-   1. T. W. Greene and P. G. M. Nuts, “Protecting Groups in Organic    Synthesis”, Second Edition (1991), John Wiley and Sons, Inc., NY;-   2. M. Schelhaas and H. Waldman, “Protecting Group Strategies in    Organic Synthesis”, Angew. Chemie Int. Ed. Engl. 35, 2056–2083    (1996);-   3. M. J. Gait, ed., “Oligonucleotide Synthesis A Practical    Approach”, IRL Press, Oxford (1984);-   4. S. A. Narang, ed., “Synthesis and Applications of DNA and RNA”,    Academic Press, Inc., Orlando (1987); and-   5. S. Agrawal, ed., “Methods in Molecular Biology, Vol. 20:    Protocols for Oligonucleotides and Analogs”, Humana Press, Totowa,    N.J. (1993);    the contents of each of which are hereby incorporated by reference,    for a discussion of other possible hydroxyl protecting groups.

The manner by which the desired nucleoside may be protected isconventional and within the purview of a person skilled in the art. See,for example U.S. Pat. No. 3,400,190 (Melby), U.S. Pat. No. 4,458,066(Caruthers et al.).

A preferred method for production of deoxyribonucleosides in the contextof the present invention is to use a nucleoside with a5′-dimethoxytrityl protecting group and an appropriate exocyclic aminoprotecting group, e.g., N⁶-benzoyl-5′-dimethoxytrityl-2′-deoxyadenosine,N⁴-benzoyl-5′-dimethoxytrityl-2′-deoxycytidine,5′-dimethoxytrityl-N²-isobutyryl-2′-deoxyguanosine, or5′-dimethoxytritylthymidine.

A preferred method for production of ribonucleosides in the context ofthe present invention is to use a 5′-dimethoxytrityl protectednucleoside, with appropriate exocyclic amino protection, and noprotecting groups on either of the 2′- or 3′-hydroxyl positions. Thelinker can then react with either one of the two adjacent hydroxylgroups (it doesn't matter which) to give a mixture of 2′- and3′-linkages. The unreacted hydroxyl groups may then be acetylated bytreatment of the immobilized nucleoside with acetic anhydride.Alternatively, ribonucleosides which have a 5′-dimethoxytrityl group,appropriate exocyclic amino group protection, and either a 3′-hydroxylprotecting group or a mixture of 2′- and 3′-protecting groups can beused. The 3′-protected compounds are generally unwanted isomers whichare simultaneously produced when the 2′-hydroxyl position is protectedand having little other use.

The reusable linker arm having the formula:Z-O-T

[SUPPORT]may be produced by a process comprising the step of reacting togetherthe compound of Formulae I and II:Z-OH   (I)HO-T

[SUPPORT]  (II)wherein Z and T are as defined above.

The reusable linker arm having the formula:NUCLEOSIDE-Z-O-T

[SUPPORT]comprises the step of reacting together the compounds of Formulae I, IIand III:HO-Z-OH   (I)HO-T

[SUPPORT]  (II)NUCLEOSIDE-OH   (III)wherein Z and T are as defined above.

The compounds of Formulae I and II or of Formulae I, II and III(depending on which version of the present linker arm is being produced)are preferably reacted in the presence of an activating agent. As usedthroughout this specification, the term “activating group” is intendedto have a broad meaning and is intended to encompass electrophilicreagents capable of activating a carboxyl moiety (e.g., on the linkingcompound of Formula II) by attachment of a leaving group to the acylcarbon of the carboxl moiety—see, for example, M. Bodanszky, “Principlesof Peptide Synthesis”, Second Edition, Springer-Verlag, Berlin (1993).Thus, the activating agent should be capable of initiating at least oneof the following: (a) formation of a reactive acylating agent (this isan example of a derivate) from the carboxyl moeiy in a separate step orsteps, followed by immediate treatment with the amino component (in thiscase, for example, an amino-terminated support) to form an amide linkageor a hydroxy component (in this case a hydroxy-terminated support or ahydroxyl group on the desired nucleoside) to form an ester linkage; (b)formation of an isolable acylating agent, separately, optionally withpurification prior to treatment with the amino or hydroxy component asdiscussed in (a); and (c) formation of an acylating intermediate in thepresence of the amino/hydroxy component, by the addition of anactivating agent to a mixture of the two components. Thus, each of (a),(b) and (c) are applicable to the formation of both carboxylic estersand amides and all three routes can be used to attach nucleosides tosupports.

For example, the Letsinger method, which first reacts oxalyl chloridewith triazole, and then adds a nucleoside to the resulting oxalyltriazolide is an example of route (a). Conversion of the carboxylic acidgroup into an “active” ester using either p-nitrophenol, or di-, tri-,tetra-, or penta-chlorinated or fluorinated phenols, orN-hydrosuccinimide are common examples of route (b). Route (c) has beenthe most commonly used method in recent years and both the carbodiimidereagents (dicyclohexylcarbodiimide,1-(3-dimethylaminopropyl)-ethylcarbodiimide, anddiisopropylcarbodiimide) and uronium reagents(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,(HBTU)) may be used in this approach.

In a preferred embodiment, in addition to an activating reagent, thereaction of the compounds of Formulae I, II and III is conducted in thepresence of a nucleophilic catalyst or additive (typically4-dimethylamino pyridine (DMAP), 1-hydroxybenzotriazole (HOBt), or1-hydroxy-7-azabenzotriazole (HOAt)) to speed up the reaction and atertiary amine base (typically triethylamine, pyridine, ordiisopropylethylamine) to ionize the carboxylic acid group.

Thus, those of skill in the art will recognize that the precise natureof the activation agent is not particularly restricted provided, ofcourse, that the activated carboxylic acid group is capable ofinitiating formation of the ester or amide linkage, as appropriate, andthe activating reagent does not have any deleterious effect on thedesired nucleoside.

Thus, activation of the carboxylic acid by conversion into an acidchloride; an active ester (i.e., nitrophenyl, nitrophenylthio,trichlorophenyl, trifluorophenyl, pentachlorophenyl, pentafluorophenyl,or 3-hydroxy-2,3-dihydro-4-oxo-benzotriazine esters); an activehydroxylamine ester (i.e., N-hydroxyphthalimide orN-hydroxysuccinimide); acid anhydride; or mixed anhydride will producederivates which will form the desired linkage, and thus, thesestrategies are encompassed herein.

Non-limiting examples of activating agents may be selected from thegroup consisting of arylsulfonyl chlorides (e.g., benzenesulfonylchloride (BS—Cl), mesitylenesulfonyl chloride (MS—Cl),triisopropylsulfonylchloride (TPS—Cl)); active arylsulfonyl esters(i.e., imidazole, triazole, nitrotriazole, or tetrazole esters of BS—Cl,MS—Cl or TPS—Cl); 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline(EEDQ); acyl carbonates; 1,1′-(carbonyldioxy)dibenzotriazoles;chlorotrimethyl-silane; carbodiimides (i.e., dicyclohexylcarbodiimide(DCC), 1-(3-dimethylaminopropyl)-ethylcarbodiimide (DEC),diisopropylcarbodiimide (DIC)) either alone or in combination withauxiliary nucleophiles (i.e., 1-hydroxybenzotriazole (HOBt),1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), or3-hydroxy-3,4-dihydro-1,2,3-benzotriazin-4-one (HOObt)) and/or catalysts(i.e., 4-dimethylaminopyridine (DMAP) or N-methylimidazole (NMI)); oruronium salts (i.e., tetramethyluronium chloride (TMU—Cl),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 2-succinimido-1,1,3,3-tetramethyluroniumtetrafluoroborate (TSTU),2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TDBTU),2-(2-oxo-1(2H)-pyridyl-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TNTU),O-(7-azabenzotriazol-1-yl)-1,3-dimethyl-1,3-dimethyleneuroniumhexa-fluorophosphate (HAMDU),O-(7-azabenzotriazol-1-yl)-1,3-dimethyl-1,3-tri-methyleneuroniumhexafluorophosphate (HAMTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uroniumhexafluorophosphate (HAPipU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uroniumhexafluorophosphate (HAPyU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU)) either alone or in combination withauxiliary nucleophiles (i.e., 1-hydroxybenzotriazole (HOBt),1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), or3-hydroxy-3,4-dihydro-1,2,3-benzotriazin-4-one (HOObt)) and/or catalysts(e.g. 4-dimethylaminopyridine (DMAP) or N-methylimidazole (NMI)) orphosphonium salts (e.g.benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazole-1-yl-oxy-trispyrrolidinophosphoniumhexafluorophosphate (PyBOP),2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate(BOI), bromo tris(pyrrolidino)phosphonium hexafluorophosphate (PyBroP),7-azabenzotriazol-1-yloxytris-(dimethylamino)phosphoniumhexafluorophosphate (AOP), and7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyAOP)) either alone or in combination withauxiliary nucleophiles and/or catalysts (discussed above) will alsoproduce the desired linkage.

Other examples of suitable activating reagents may be found in any ofthe following references:

-   M. Bodanszky, “Principles of Peptide Synthesis”, Second Edition,    Springer-Verlag, Berlin (1993);-   J. Jones, “Amino Acid and Peptide Synthesis”, Oxford University    Press, Oxford (1992);-   G Grant, “Synthetic Peptides: A Users Guide”, W. H. Freeman & Co.,    NY (1992);-   E. Haslam, Tetrahedron, 36, pg. 2409, (1980); and-   M. A. Ogliaruso and J. F. Wolfe, “Synthesis of Carboxylic Acids,    Esters and Their Derivatives”, John Wiley & Sons, Chicester (1991);    the contents of each of which are hereby incorporated by reference.

In producing the present linker arm, the order of reaction is notparticularly restricted. Thus, in one embodiment (this is the preferredembodiment), the compounds of Formulae I and III are initially reactedto form a conjugate which is reacted with the compound of Formula II. Inanother embodiment, the compounds of Formulae I and II are initiallyreacted to form a conjugate which is reacted with the compound ofFormula III.

The addition of compounds of Formulae I and III to Formula II, usuallywill not result in the quantitative conversion of each immobilizedhydroxyl group into a derivatized ligand. Therefore, it is preferredthat unreacted hydroxyl groups on the surface of the support beprotected (capped) by reaction with a capping reagent. This willmitigate the free hydroxyl group participating in subsequentoligonucleotide chain extension reactions, resulting in defect sequenceslacking the terminal nucleoside. Preferably, the capping reagent shouldbe reversible so that the capping agent can be removed to regenerate thehydroxyl sites prior to the next round of support derivatization.Capping of the unreacted sites is conventional and can be performed byreaction with an activated carboxylic acid or anhydride to form anester, or by addition of a protecting group, as described hereinabove.Thus, for example, t-butylphenoxyacetic anhydride, methoxyaceticanhydride or preferably chloroacetic anhydride, combined with2,6-lutidine and N-methylimidazole in THF solution are useful examplesof capping reagents.

With reference to FIG. 2 there is illustrated a preferred pathwayillustrating the use of the present linker arm in a recycled/regeneratedmanner. In FIG. 2, DMT refers to dimethoxytrityl and B refers to anucleobase as described hereinabove. As will be apparent to those ofskill in the art, the support is recycled after oligonucleotide cleavageand support regeneration to a point in the reaction scheme where it mayagain be coupled with the HQPD-nucleoside conjugate for furtheroligonucleotide synthesis.

With further reference to “Oligo Synthesis” (Step #3) in FIG. 2, oncethe present linker arm has been produced, it may be used in theconventional manner to synthesize an oligonucleotide—see, for example,U.S. Pat. No. 5,112,962 (Letsinger). Once the oligonucleotide has beensynthesized, it may be cleaved from the solid support to yield the freeoligonucleotide and the support may then be regenerated—see Step #4 ofFIG. 2.

The cleavage step comprises hydrolysis at the point of attachment of theinitial nucleoside to the linking compound. The regeneration of thesupport involves the removal of two moieties: (i) the removal of thestructure represented by Formula I (above) from Formula II (above),which occurs simultaneously with the release of the oligonucleotideproduct, and (ii) the removal of the moiety used to protect (cap)unreacted hydroxyl sites of Formula II (above) on the support. Removalof these two moieties can occur simultaneously or separately toregenerate the support. Simultaneous removal of both moieties using onlya single reagent is simpler but care should be taken to use reagentswhich will not deleteriously affect the oligonucleotide product. Atwo-step regeneration involving the removal of the oligonucleotide usingone reagent (typically ammonium hydroxide) and then treatment of thesupport with a second reagent (which may be faster but otherwisedamaging to the oligonucleotide product thereby necessitating use of atwo-step regeneration) allows flexibility in the choice of capping andregeneration reagents.

The reagent used to effect cleavage is not particularly restricted andis within the purview of a person skilled in the art. Preferably, thereagent is a base mild enough not to damage the oligonucleotide productbut sufficiently strong to effect rapid cleavage. Non-limiting examplesof suitable reagents for this purpose may be selected from the groupconsisting of ammonium hydroxide, ammonium hydroxide/methanol,ammonia/methanol, ammonium hydroxide/methylamine, potassiumcarbonate/methanol, t-butylamine, ethylenediamine, methylamine,dimethylamine, trimethylamine/water and the like. Cleavage may also beperformed under neutral conditions using fluoride ion (i.e. 1Mtetrabutylammonium fluoride/THF or triethylamine trihydrofluoride). Thereagent used to remove the capping reagent from unreacted sites mayconsist of the above reagents or other stronger bases such as sodium orpotassium hydroxide. In our preferred embodiment, ammonium hydroxide canbe used to cleave the oligonucleotide product from the support, removethe HQPD linker arm, and cleave chloroacetyl protected hydroxyl groupsin a single regeneration step. The preferred temperature for thecleavage and regeneration is room temperature, but higher or lowertemperatures can be employed, subject to the limitations of theapparatus used.

With reference to FIG. 3, there are illustrated specific preferredexamples of hydroxyl reusuable linker arms falling within the scope ofthe present invention.

Embodiments of the invention will be illustrated in the followingExamples which should not be construed as limiting the scope of theinvention. In the Examples, the following materials were used:

1. Long chain alkylamine (LCAA) or glycerol (Gly) derivatized controlledpore glass (CPG) beads (120/200 mesh) were obtained from CPG Inc(Lincoln Park, N.J.);

2. Toyopearl AF-amino-650M and HW65F supports were obtained fromTosoHaas (Montgomeryville, Pa.);

3. Other supports were obtained from the manufacturers listed in Tables1 and 2;

4. HQPD. Hydroquinone-O,O′-diacetic acid, commercially available fromLancaster Synthesis Ltd. (Lancashire, England);

5. Ammonium hydroxide solutions (28–30%) and solvents were obtained fromVWR Canlab (Edmonton, Alberta, Canada);

6. Capping solutions were formulated as either Cap A (aceticanhydride/2,6-lutidine/THF in a volume ratio of 1:1:8) and Cap B(N-methylimidazole and THF in a volume ratio of 16:84) or Cap A(chloroacetic anhydride and THF, 17% by weight) and Cap B (2,6-lutidineand N-methylimidazole in THF in a volume ratio of 12:16:72);

7. Anhydrous pyridine and acetonitrile, distilled from CaH₂;

8. DIEA, diisopropylethylamine, reagent grade;

9. MeCN, acetonitrile, low water DNA synthesis grade;

10. DMAP, 4-dimethylaminopyridine, reagent grade;

11. DEC, 1-(3-dimethylaminopropyl)-ethylcarbodiimide, reagent grade;

12. Sulfurizing reagent, Beaucage thiolating reagent, from PharmaciaBiotech, was used as a 0.05M solution in acetonitrile; and

13. HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate, reagent grade;

In the following Examples the amount of nucleoside (loading) on theinsoluble supports was determined by spectrophotometric trityl analysis.In this procedure, a sample of support (4–5 mg) was accurately weigheddirectly into a 10 mL volumetric flask. A solution of dichloroaceticacid in 1,2-dichloroethane in a volume ration of 5:95 was then added tofill the flask. The contents were then thoroughly mixed and theabsorbance of the orange coloured solution was measured at 503 nm usinga Philips UV/Vis spectrophotometer. The nucleoside loading (in μmol/g ofCPG) was then calculated as:Loading=(A ₅₀₃ ×Vol×1000)/(Wt×76)wherein A₅₀₃=absorbance at 503 nm, Vol=solution volume in mL, andWt=amount of CPG tested in mg. The accuracy of the trityl determinationwas approximately ±2–3%.

EXAMPLE 1 Synthesis of Nucleoside-3′-O-Hoda Hemiesters

5′-Dimethoxytrityl-N-protected deoxyribonucleoside (10 mmol),hydroquinone-O,O′-diacetic acid (15 mmol, 3.39 g),4-dimethylaminopyridine (1 mmol, 122 mg), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15 mmol,2.88 g) were combined in a 100 mL round bottom flask equipped with amagnetic stir bar. Triethylamine (0.8 mL) and anhydrous pyridine (50 mL)were added to the flask and the contents were stirred at roomtemperature overnight.

The reaction was checked by TLC (5% methanol/chloroform). If more than atrace of starting nucleoside was visible, more1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2–5 mmol)was added to the reaction and stirring was continued for another day.When TLC showed complete disappearance of the starting nucleoside, thesolution was concentrated by evaporation until a thick oil was formed.The oil was redissolved in chloroform (˜200 mL) and transfer to aseparatory funnel. The chloroform solution was washed with aqueoussodium bicarbonate (˜100 mL×2) and then water (˜100 mL×3). The funnelwas slowly inverted to mix the two phases. The chloroform phase wascollected and the aqueous phase was discarded. If an inseparableemulsion was formed, then either centrifugation (for small volumes) or(for large volumes) precipitation by addition of hexanes followed byfiltration and redissolving the sticky precipitate back into chloroformcan be performed.

The chloroform solution was added to anhydrous magnesium sulfate andmixed to remove residual moisture from the solution. The magnesiumsulfate was filtered off, the filtrated was washed with a small amountof chloroform and then the chloroform solution was evaporated todryness. A light brown foam, containing a mixture of diester andnucleoside hemiester sodium salt was formed and solidified.

The hemiester sodium salt was converted into a more soluble pyridiniumsalt by dissolving the foam in pyridine (˜50–100 mL) and then adding AG50W-X4H⁺ cation exchange resin (2 eq.). The mixture was stirred forapproximately 5 minutes and then the ion exchange resin was filteredoff. The pyridine solution was evaporated to dryness. A light brown foamformed and solidified. The sold was dried under vacuum overnight toremove excess pyridine.

EXAMPLE 2 Preparation of 12-Dimethoxytrityl-Hydroxydo-Decanoic AcidDerivatized Supports

This example describes the synthesis of a C₁₂ linker arm within thescope of the present invention and how it can be used to convertcommercially available amino-derivatized supports into reusablehydroxyl-derivatized supports.

12-Hydroxydodecanoic acid (9.25 mmol) was coevaporated to dryness withpyridine (3×). Then pyridine (˜40 mL) and dimethoxytrityl chloride (10.2mmol) were added. After stirring overnight, the solution wasconcentrated (to 10 mL), diluted with CHCl₃ (50 mL), washed with aq.NH₄HCO₃ (3×) and water (2×). The crude material was then purified on asilica gel column by elution with a 1% TEA/CHCl₃-4% MeOH/1% TEA/CHCl₃gradient. The prodcut yield was 6.7 mmol (72%) of12-dimethoxytrityl-hydroxydodecanoic acid as a brown oil.

An amino functionalized support (0.5 g),12-dimethoxytritylhydroxy-dodecanoic acid (0.2 mmol),4-dimethylaminopyridine (0.1 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.6 mmol),triethylamine (0.1 mL), and pyridine (7 mL) were shaken at roomtemperature (16 h). The support was filtered off, washed, and dried.Linker loading was determined by trityl analysis and the results areprovided in Table 1. Unreacted amino and hydroxyl groups on thederivatized support (if present) were then acetylated by treating thesupport with equal volumes 1 M acetic anhydride/2,6-lutidine/THF (Cap A)and 2M N-methylimidazole/THF (Cap B) reagents for 3 hours. The supportwas then filtered off, washed, and dried.

TABLE 1 Loading Results Using 12-Dimethoxytrityl- hydroxydodecanoic AcidLinker Arm Linker arm Experiment Support loading (μmol/g) 1 PharmaciaHL-30 amino primer support 216 2 Long chain alkylamine CPG 87 3 AminoTentagel, Millipore 107 4 Toyopearl AF-amino-650M 217 5 Aminoethylpolystyrene, Hamilton 73 6 Aminomethyl polystyrene, 28 AppliedBiosystems

EXAMPLE 3 Derivatization of Toyopearl HW-65F Support with 1,4-ButanediolDiglycidyl Ether

This Example describes how hydroxyl surface groups on commerciallyavailable Toyopearl HW65 supports are extended with a butane diglycidyllinker to create a reusable support.

Toyopearl HW-65F vinyl alcohol/methacrylic acid copolymer was obtainedas a slurry in 500 ml 20% ethanol/water. This slurry was evaporated todryness to yield of 90 g of dry support. The hydroxyl content of the drysupport was determined, in triplicate, by derivatization withdimethoxytrityl chloride/tetrabutylammonium perchlorate and tritylanalysis, to be 1,095 μmol/g.

The dry HW-65F support (25 g), 1.0 M aqueous NaOH solution containing 1mg/mL NaBH₄ (100 mL) and 1,4-butanediol diglycidyl ether (75 mL) wereshaken at room temperature (3.5 h). The support was filtered off andwashed with water, acetonitrile, and then chloroform. After drying, DMTderivatization and analysis (M. P. Reddy and P. J. Voelker, 1988, Int.J. Peptide Protein Res. 31, 345–348,) of a sample indicated 902 μmol/gof remaining hydroxyl groups. Therefore, the epoxide loading wasestimated to be 193 μmol/g.

The epoxide derivatized support (25 g), benzoic anhydride (51 g),4-dimethylaminopyridine (6.6 g) and anhydrous pyridine (180 mL) wereshaken at room temperature (overnight) to benzoylate unreacted hydroxylgroups. The support was filtered off, washed (methanol, thenchloroform), and dried. DMT derivatization and analysis indicated thatthe residual hydroxyl group loading had decreased to only 5 μmol/g.

The benzoylated support (25 g), THF (140 mL), and 2.9 N aqueous HClO₄(16.6 mL, 48 mmol) were shaken at room temperature (13 h). Tritylderivatization and analysis of an aliquot showed an hydroxyl loading of98 μmol/g. Additional 2.9 N HClO₄ (34 mL) was added and shakingcontinued for another 3 h. The support was filtered off, washed, anddried and a final trityl derivatization and analysis indicated anhydroxyl loading of 103 μmol/g.

EXAMPLE 4 Synthesis of Oligonucleotide Phosphorothioates and SupportRecycling Using Chloroacetic Anhydride Capping

This Example provides experiments which illustrate the suitability of avariety of different supports for repetitive oligonucleotide synthesis.

The following reagents were installed on a Perkin-Elmer/AppliedBiosystems 394 4-column, 8-base position DNA synthesizer:

-   Ports #1–4: dA^(Bz), dG^(iBu), dC^(Bz), and T phosphoramidites (0.2    M solutions).-   Port #7: 0.15 M    5′-dimethoxytrity-N⁶-benzoyl-2′-deoxyadenosine-3′-O-hydro-quinone-O,O′-diacetyl    hemiester pyridinium salt and 0.15 M diisopropylethylamine in    anhydrous acetonitrile.-   Port #8: 0.15 M HBTU and 0.15M DMAP in anhydrous acetonitrile.-   Port #9: 0.45 M Tetrazole/acetonitrile.-   Port #10: 28% Ammonium hydroxide.-   Port #11: 1 M Chloroacetic anhydride in THF (Cap A reagent).-   Port #12: 1 M 2,6-Lutidine and 2 M N-methylimidazole in THF (Cap B    reagent).-   Port #14: 5% (v/v) Dichloroacetic acid/1,2-dichloroethane.-   Port # 15: 0.05 M Beaucage reagent in acetonitrile.

Up to four synthesis columns, each containing one of the supports listedin Table 2, were installed on the synthesizer and, if necessary,manually detritylated to deblock the hydroxyl linker arm.

The synthesizer was then programmed to automatically execute thefollowing steps:

-   -   1: A “Begin” procedure consisting of a column wash, nucleoside        coupling to the support by simultaneous addition (4.0 sec) of        nucleoside hemiester (port #7) and coupling reagent (port #8)        and a 600 sec wait, column wash, capping of unreacted hydroxyl        sites (Cap A+B reagents, 300 sec), column wash, and priming of        ports #1, 2, 3, 4, and 9.    -   2: Synthesis of the 20-base phosphorothioate oligonucleotide        sequence dGCCCAAGCTGGCATCCGTCA (trityl-off) (Sequence ID No. 1).    -   3: A 15 minute ammonium hydroxide hydrolysis step to cleave the        oligonucleotide from the support.

After completion of the ammonium hydroxide hydrolysis, the columns wereremoved from the synthesizer, manually treated with 0.05 M potassiumcarbonate/methanol solution (5 min), rinsed with methanol, dried byaspiration (5 min), re-installed on the synthesizer, and rinsed withanhydrous acetonitrile. The automated synthesis was then repeated (i.e.,Steps 1, 2, and 3 above) using the same synthesis column a total oftwelve times.

The amount of trityl color released after the first detritylation stepwas collected and quantitated to determine the amount of nucleosideadded to the support—the results are reported in Table 3. The releasedoligonucleotide solution was deprotected (55° C. 16 h), evaporated toremoved ammonia, and quantitated by UV at 260 nm—the results arereported in Table 4. The correct identity of the products, obtained fromeach of the results shown Table 4, was verified by electrophoresis andcomparison to authentic material. Furthermore, no unusual impurities,attributable to the support recycling were present. These resultsconfirmed that each of the nine supports used in this experiment couldbe reused and in several cases satisfactory results (comparable to newsupports) were obtained, even after six or more uses.

EXAMPLE 5 Synthesis of Oligonucleotide Phosphorothioates and SupportRecycling Using Methoxyacetic Anhydride Capping

This Example illustrates the use of methoxyacetic anhydride as thecapping reagent instead of chloroacetic anhydride used in the previousExamples.

The automated DNA synthesizer was set-up with reagents, as described inExample 4, with the exception of the Cap A and B reagents, which were asfollows:

-   Port #10: 0.5 M Methoxyacetic anhydride and 0.5 M 2,6-lutidine in    acetonitrile (Cap A).-   Port #12: 1 M N-Methylimidazole in acetonitrile (Cap B).

The automated nucleoside derivatization, oligonucleotide synthesis, andsupport recycling procedure was then performed using the supports listedin Table 5 and the procedure described in Example 4. However, because ofthe greater stability of the methoxyacetyl group, the manual columnregeneration step with 0.05M potassium carbonate/methanol was increasedfrom 5 min to 15 min.

The amount of trityl color released after the first detritylation stepwas collected and quantitated to determine the amount of nucleosideadded to the support—the results are reported in Table 6. The releasedoligonucleotide solution was deprotected (55° C., 16 h), evaporated toremoved ammonia, and quantitated by UV at 260 nm—the results arereported in Table 7. The composition of the products obtained in Table 7was examined by gel electrophoresis and the expected products wereobtained in each case. This indicated that methoxyacetic anhydride couldalso be used as a satisfactory capping reagent during the supportrecycling.

TABLE 2 Supports Used For Phosphorothioate Synthesis and SupportRecycling Experiment Support Linker Arm Amount used (mg) 1 Long chainalkylamine CPG Hydroxyhexylsuccinyldiamide 16.2 2 Long chain alkylamineCPG Hydroxydodecanoic acid 21.1 3 Glycerol CPG 13.3 4 ToyopearlAF-amino-650M Hydroxydodecanoic acid 15 5 Aminoethyl polystyrene,Hamilton Hydroxydodecanoic acid 21.5 6 Aminomethyl polystyrene, AppliedBiosystems Hydroxydodecanoic acid 34.1 7 Pharmacia hydroxyl primersupport* Butanediol diglycidyl 14.5 8 Toyopearl HW65F Butanedioldiglycidyl 15.4 9 Hydroxyethyl polymethacrylate/polystyrene, Hamilton27.3 *proprietary material supplied by Pharmacia

TABLE 3 Nucleoside loading obtained after repetitive synthesis on thesame support Synthesis # and First Nucleoside Loading Level (μmol/g)Experiment 1 2 3 4 5 6 7 8 9 10 11 12 1 74 62 61 65 56 55 54 60 58 60 5149 2 57 58 54 56 54 54 54 62 51 49 56 46 3 72 68 65 67 64 64 62 61 60 5755 54 4 96 119 130 130 126 123 65 60 46 34 25 17 5 75 79 76 76 70 57 4740 43 60 46 40 6 30 35 35 37 38 35 35 36 31 33 37 33 7 155 111 107 97122 115 95 122 87 110 101 89 8 107 110 116 113 111 104 102 87 76 92 7166 9 34 41 47 48 48 53 49 50 45 62 51 67

TABLE 4 Amount of Crude Oligonucleotide Produced From RepetitiveSyntheses on the Same Support Synthesis # and amount of crude productproduced (A₂₆₀ units) Experiment 1 2 3 4 5 6 7 8 9 10 11 12 1 7530 75907530 7650 6790 6850 5740 5930 4750 3640 2780 2350 2 7630 7680 6590 69706680 6680 6110 5170 3460 2420 2700 1850 3 8350 7740 8270 7970 8120 81207440 7370 6690 6170 5190 4660 4 12200 11500 12600 11700 11600 11800 98806710 5850 4750 3540 2550 5 8010 8340 7990 7960 7810 6720 5690 3900 32803780 2550 2050 6 3610 4300 4220 4540 4830 4560 4630 4620 4300 3730 33902750 7 9300 8190 7460 7620 8550 7390 5900 4900 2280 4180 2430 1750 811520 11100 10600 11400 11500 11400 9980 n.d. n.d. 8390 5060 6270 9 23603260 4520 4630 5290 6420 6610 n.d. n.d. 8570 6420 6830

TABLE 5 Supports Used For Oligonucleotide Phosphorothioate Synthesis andSupport Recycling Experiment Support Linker Arm Amount used (mg) 1Glycerol CPG 14.5 2 Toyopearl HW65F Butanediol 14.6 diglycidyl

TABLE 6 Nucleoside Loading Obtained After Repetitive Synthesis on theSame Support Synthesis # and first nucleoside loading (μmol/g)Experiment 1 2 3 4 5 6 7 8 9 10 11 12 1 83 82 71 75 61 69 63 64 58 56 4842 2 93 105 115 115 105 114 117 120 110 113 96 67

TABLE 7 Amount of Crude Oligonucleotide Produced From RepetitiveSyntheses on the Same Support Synthesis # and amount of crude productproduced (A₂₆₀ units) Experiment 1 2 3 4 5 6 7 8 9 10 11 12 1 8100 77507570 7850 6910 7500 7440 7400 6870 6810 6210 5220 2 8480 9820 1020010900 9490 10200 10300 10700 10000 9190 8450 6170

1. A reusable linker arm for solid support oligonucleotide synthesis,the linker arm consisting of the following formula:Z-O-T

[SUPPORT] wherein Z is selected from the group consisting of:HO₂C—CH₂—CH₂—(C═O)—; HO₂C—CH₂—O—CH₂—(C═O)—; HO₂C—(C═O)—; and

wherein: R¹, R² and R³ are the same or different and are selected fromthe group consisting of hydrogen, halide, a substituted or unsubstitutedC₁–C₂₀ alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl groupand a substituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁴ and R⁵ arethe same or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; X¹ is selected from the groupconsisting of —O—, —S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected fromthe group consisting of hydrogen, a substituted or unsubstituted C₁–C₂₀alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; n is 0, 1 or 2; andone of A¹ and B¹ is selected from the group consisting of hydrogen,halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group and the other of A¹ and B¹ has theformula:

wherein p is 0 or 1; X² is selected from the group consisting of —O—,—S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected from the groupconsisting of hydrogen, a substituted or unsubstituted C₁–C₂₀ alkylgroup, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁶ and R⁷ are thesame or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; and T has the formula:—[CH₂]_(q)—[O—CH₂—CH₂—O]_(r)—[CH₂]_(s)— wherein q and s are the same ordifferent and each is an integer having a value of 0–40 and r is aninteger having a value of 1–200 or T has the formula:-[Q]_(a)-CH₂—CH(R^(a))—CH₂—O—[CH₂]_(b)— wherein a is 0 or 1, R^(a) isselected from —OH, —NH₂, —NHR and —OR wherein R is a protecting groupand b is an integer having a value of 0–40, and Q is moiety having theformula:—[CH₂]_(u)—[CH(R^(a′))]_(t)—[CH₂]_(q)—O—[CH₂]_(r)—O—[CH₂]_(s)— whereinq, r, s, t and u are the same or different and each is an integer havinga value of 0–40 and R^(a′) is selected from the group consisting ofhydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, a C₁–C₄₀alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxy-containing group, aC₂–C₄₀ acrylate-containing group, a C₅–C₄₀ alkylaryl group, —NH₂, —NHRand —OR, wherein R is a protecting group; and wherein the term SUPPORTis defined as an organic or an inorganic substance.
 2. The reusablelinker arm defined in claim 1, wherein T has the formula:

wherein q and s are the same or different and each is an integer havinga value of 0–40 and r is an integer having a value of 1–200.
 3. Thereusable linker arm defined in claim 2, wherein q and s are the same ordifferent and each is an integer having a value of 1–20 and r is aninteger having a value of 1–150.
 4. The reusable linker arm defined inclaim 1, wherein T has the formula:

wherein a is 0 or 1, R^(a) is selected from —OH, —NH₂, —NR and —ORwherein R is a protecting group and b is an integer having a value of0–40, and Q is a moiety having the formula:

wherein q, r, s, t and u are the same or different and each is aninteger having a value of 0–40 and R^(a′) is selected from the groupconsisting of hydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ arylgroup, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀hydroxy-containing group, a C₂–C₄₀ acrylate-containing group, a C₅–C₄₀alkylaryl group, —NH₂, —NHR and —OR, wherein R is a protecting group. 5.The reusable linker arm defined in claim 4, wherein a is 0 and R^(a) is—OH.
 6. The reusable linker arm defined in claim 4, wherein a is 1 andR^(a) is —NR or —OR.
 7. The reusable linker arm defined in claim 4,wherein the protecting group is selected from the group consisting ofacetyl, chloroacetyl, methoxyacetyl, t-butyl phenoxyacetyl,phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT),pivalyl-isobutyloxycarbonyl, t-butyldimethylsilyl, 9-phenylxanthen-9-yl(pixyl), tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl,benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl,dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl,isopropyl-dimethylsilyl, diisopropylmethylsilyl, diethylisopropylsilyl,triisopropylsilyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl,o-nitrobenzyl, o-hydroxystyryldimethylsilyl, 2-oxo-1,2-diphenylethyl,allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl,fluorenyl-methoxycarbonyl, 2-phenylsulfonyl-ethoxycarbony,fluorophenyl-methoxypiperidinyl and mixtures thereof.
 8. The reusablelinker arm defined in claim 1, wherein s is 0, q, r and u are the sameor different and each is an integer having a value of 1–10, t is aninteger of 1–5 and R^(a) is hydroxyl.
 9. The reusable linker arm definedin claim 1, wherein T has the formula:

wherein a is 0 or 1, R^(a) is selected from —OH, —NH₂, —NR and —ORwherein R is a protecting group and b is an integer having a value of0–40.
 10. The reusable linker arm defined in claim 9, wherein a is 0 andR^(a) is —OH.
 11. The reusable linker arm defined in claim 9, wherein ais 1 and R^(a) is —NR or —OR.
 12. The reusable linker arm defined inclaim 4, wherein the moiety Q is unsubstituted.
 13. The reusable linkerarm defined in claim 4, wherein the organic moiety is substituted by atleast one moiety selected from the group consisting of a C₁–C₄₀ alkylgroup, a C₅–C₄₀ aryl group, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group,a C₁–C₄₀ hydroxy-containing group, a C₂–C₄₀ acrylate-containing groupand a C₅–C₄₀ alkylaryl group.
 14. The reusable linker arm defined inclaim 4 wherein Q is additionally defined as having the formula:—[CH₂]_(x)—C(═O)—NH—[CH₂]_(y)—NH—C(═O)—[CH₂]_(z)— wherein each of x, yand z is an integer having a value of 1–40.
 15. The reusable linker armdefined in claim 1, wherein p is
 0. 16. The reusable linker arm definedin claim 1, wherein B¹ is selected from the group consisting ofhydrogen, halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, asubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group.
 17. The reusable linker armdefined in claim 1, wherein each of R⁴, R⁵, R⁶ and R⁷ is hydrogen. 18.The reusable linker arm defined in claim 1, wherein each of m and nare
 1. 19. The reusable linker arm defined in claim 1, wherein each ofR¹, R² and R³ is hydrogen.
 20. The reusable linker arm defined in claim1, wherein X¹ and X² are both —O—.
 21. The reusable linker arm definedin claim 1, wherein SUPPORT is an inorganic substance.
 22. The reusablelinker arm defined in claim 21, wherein the inorganic substance isselected from the group consisting of silica, glass beads, porous glass,aluminosilicates, borosilicates, metal oxides, clays and mixturesthereof.
 23. The reusable linker arm defined in claim 1, wherein SUPPORTis an organic substance.
 24. The reusable linker arm defined in claim23, wherein the organic substance is a cross-linked polymer.
 25. Areusable linker arm for solid support oligonucleotide synthesisconsisting of the following formula:NUCLEOSIDE-Z-O-T

[SUPPORT] wherein Z is selected from the group consisting ofHO₂C—CH₂—CH₂—(C═O)—; HO₂C—CH₂—O—CH₂—(C═O)—; HO₂C—(C═O)—; and

wherein: R¹, R² and R³ are the same or different and are selected fromthe group consisting of hydrogen, halide, a substituted or unsubstitutedC₁–C₂₀ alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl groupand a substituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁴ and R⁵ arethe same or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; X¹ is selected from the groupconsisting of —O—, —S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected fromthe group consisting of hydrogen, a substituted or unsubstituted C₁–C₂₀alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; n is 0, 1 or 2; andone of A¹ and B¹ is selected from the group consisting of hydrogen,halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group and the other of A¹ and B ¹ has theformula:

wherein p is 0 or 1; X² is selected from the group consisting of —O—,—S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected from the groupconsisting of hydrogen, a substituted or unsubstituted C₁–C₂₀ alkylgroup, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁶ and R⁷ are thesame or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; and T has the formula:—[CH₂]_(q)—[O—CH₂—CH₂—O]_(r)—[CH₂]_(s)— wherein q and s are the same ordifferent and each is an integer having a value of 0–40 and r is aninteger having a value of 1–200 or T has the formula:-[Q]_(a)-CH₂—CH(R^(a))—CH₂—O—[CH₂]_(b)— wherein a is 0 or 1, R^(a) isselected from —OH, —NH₂, —NHR and —OR wherein R is a protecting groupand b is an integer having a value of 0–40, and Q is moiety having theformula:—[CH₂]_(u)—[CH(R^(a′))]_(t)—[CH₂]_(q)—O—[CH₂]_(r)—O—[CH₂]_(s)— whereinq, r, s, t and u are the same or different and each is an integer havinga value of 0–40 and R^(a′) is selected from the group consisting ofhydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, a C₁–C₄₀alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxy-containing group, aC₂–C₄₀ acrylate-containing group, a C₅–C₄₀ alkylaryl group, —NH₂, —NHRand —OR, wherein R is a protecting group; and wherein the term SUPPORTis defined as an organic or an inorganic substance and wherein the termNUCLEOSIDE represents an optionally protected ribonucleosidyl or2′-deoxyribonucleosidyl group.
 26. The reusable linker arm defined inclaim 25, wherein T has the formula:

wherein q and s are the same or different and each is an integer havinga value of 0–40 and r is an integer having a value of 1–200.
 27. Thereusable linker arm defined in claim 26, wherein q and s are the same ordifferent and each is an integer having a value of 1–20 and r is aninteger having a value of 1–150.
 28. The reusable linker arm defined inclaim 25, wherein T has the formula:

wherein a is 0 or 1, R^(a) is selected from —OH, —NH₂, —NR and —ORwherein R is a protecting group and b is an integer having a value of0–40, and Q is a moiety having the formula:

wherein q, r, s, t and u are the same or different and each is aninteger having a value of 0–40 and R^(a′) is selected from the groupconsisting of hydrogen, hydroxyl a C₁–C₄₀ alkyl group, a C₅–C₄₀ arylgroup, a C₁–C₄₀ alkoxy group a C₁–C₄₀ ester group a C₁–C₄₀hydroxy-containing group a C₂–C₄₀ acrylate-containing group, a C₅–C₄₀alkylaryl group, —NH₂, —NHR and —OR, wherein R is a protecting group.29. The reusable linker arm defined in claim 28, wherein a is 0 andR^(a) is —OH.
 30. The reusable linker arm defined in claim 28, wherein ais 1 and R^(a) is —NR or —OR.
 31. The reusable linker arm defined inclaim 28, wherein the protecting group is selected from the groupconsisting of acetyl, chloroacetyl, methoxyacetyl, t-butylphenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT),pivalyl-isobutyloxycarbonyl, t-butyldimethylsilyl, phenoxyacetal,9-phenylxanthen-9-yl (pixyl), tetrahydropyranyl,methoxytetrahydropyranyl, methoxymethyl, benzyloxymethyl,methoxyethoxymethyl, methylthiomethyl, dialkylphosphate, levulinyl,dimethylphenylsilyl, trimethylsilyl, isopropyl-dimethylsilyl,diisopropylmethylsilyl, diethylisopropylsilyl, triisopropylsilyl,benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl, o-nitrobenzyl,o-hydroxystyryldimethylsilyl, 2-oxo-1,2-diphenylethyl, allyloxycarbonyl,monomethoxymethyl, nitroveratryloxycarbonyl, fluorenyl-methoxycarbonyl,2-phenylsulfonyl-ethoxycarbony, fluorophenyl-methoxypiperidinyl andmixtures thereof.
 32. The reusable linker arm defined in claim 28,wherein s is 0, q, r and u are the same or different and each is aninteger having a value of 1–10, t is an integer of 1–5 and R^(a) ishydroxyl.
 33. The reusable linker arm defined in claim 28, wherein T hasthe formula:

wherein a is 0 or 1, R^(a) is selected from —OH, —NH₂, —NR and —ORwherein R is a protecting group and b is an integer having a value of0–40, and Q is a moiety having the formula:

wherein q, r, s, t and u are the same or different and each is aninteger having a value of 0–40 and R^(a′) is selected from the groupconsisting of hydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ arylgroup, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group hydroxy-containinggroup, a C₂–C₄₀ acrylate-containing group, a C₅–C₄₀ alkylaryl group,—NH₂, —NHR and —OR, wherein R is a protecting group.
 34. The reusablelinker arm defined in claim 33, wherein a is 0 and R^(a) is —OH.
 35. Thereusable linker arm defined in claim 33, wherein a is 1 and R^(a) is—NHR or —OR.
 36. The reusable linker arm defined in claim 33, whereinthe organic moiety is unsubstituted.
 37. The reusable linker arm definedin claim 33, wherein the moiety Q is substituted by at least one moietyselected from the group consisting of a C₁–C₄₀ alkyl group, a C₅–C₄₀aryl group, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀hydroxy group, a C₂–C₄₀ acrylate group and a C₅–C₄₀ alkylaryl group. 38.The reusable linker arm defined in claim 4 wherein Q is additionallydefined as having the formula:—[CH₂]_(x)—C(═O)—NH—[CH₂]_(y)—NH—C(═O)—[CH₂]_(z)— wherein each of x, yand z is an integer having a value of 1–40.
 39. The reusable linker armdefined in claim 25, wherein p is
 0. 40. The reusable linker arm definedin claim 25, wherein B¹ is selected from the group consisting ofhydrogen, halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, asubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group.
 41. The reusable linker armdefined in claim 25, wherein each of R⁴, R⁵, R⁶ and R⁷ is hydrogen. 42.The reusable linker arm defined in claim 25, wherein each of m and nare
 1. 43. The reusable linker arm defined in claim 25, wherein each ofR¹, R² and R³ is hydrogen.
 44. The reusable linker arm defined in claim25, wherein X¹ and X² are both —O—.
 45. The reusable linker arm definedin claim 25, wherein SUPPORT is an inorganic substance.
 46. The reusablelinker arm defined in claim 45, wherein the inorganic substance isselected from the group consisting of silica, glass beads, porous glass,aluminosilicates, borosilicates, metal oxides, clays and mixturesthereof.
 47. The reusable linker arm defined in claim 25, whereinSUPPORT is an organic substance.
 48. The reusable linker arm defined inclaim 47, wherein the organic substance is a cross-linked polymer. 49.The reusable linker arm defined in claim 25, wherein NUCLEOSIDE is amoiety selected from one of the following formulae:

wherein R⁸ and R¹⁰ are hydrogen or a protecting group, R⁹ is hydrogen or—OR¹¹ wherein R¹¹ protecting group, and B* is a nucleic acid base.
 50. Aprocess for production of a reusable linker arm for oligonucleotidesynthesis having the following formula:Z-O-T

[SUPPORT] wherein Z is selected from the group consisting of:HO₂C—CH₂—CH₂—(C═O)—; HO₂C—CH₂—O—CH₂—(C═O)—; HO₂C—(C═O)—; and

wherein: R¹, R² and R³ are the same or different and are selected fromthe group consisting of hydrogen, halide, a substituted or unsubstitutedC₁–C₂₀ alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl groupand a substituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁴ and R⁵ arethe same or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; X¹ is selected from the groupconsisting of —O—, —S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected fromthe group consisting of hydrogen, a substituted or unsubstituted C₁–C₂₀alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; n is 0, 1 or 2; andone of A¹ and B ¹ is selected from the group consisting of hydrogen,halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group and the other of A¹ and B¹ has theformula:

wherein p is 0 or 1; X² is selected from the group consisting of —O—,—S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected from the groupconsisting of hydrogen, a substituted or unsubstituted C₁–C₂₀ alkylgroup, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁶ and R⁷ are thesame or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; and T has the formula:—[CH₂]_(q)—[O—CH₂—CH₂—O]_(r)—[CH₂]_(s)— wherein q and s are the same ordifferent and each is an integer having a value of 0–40 and r is aninteger having a value of 1–200 or T has the formula:-[Q]_(a)-CH₂—CH(R^(a))—CH₂—O—[CH₂]_(b)— wherein a is 0 or 1, R^(a) isselected from —OH, —NH₂, —NHR and —OR wherein R is a protecting groupand b is an integer having a value of 0–40, and Q is moiety having theformula:—[CH₂]_(u)—[CH(R^(a′))]_(t)—[CH₂]_(q)—O—[CH₂]_(r)—O—[CH₂]_(s)— whereinq, r, s, t and u are the same or different and each is an integer havinga value of 0–40 and R^(a′) is selected from the group consisting ofhydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, a C₁–C₄₀alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxy-containing group, aC₂–C₄₀ acrylate-containing group, a C₅–C₄₀ alkylaryl group, —NH₂, —NHRand —OR, wherein R is a protecting group, the process comprising asequence of steps or reacting together the compounds of the Formulae Iand IIHO-Z-OH  (I)HO-T

[SUPPORT]  (II) in the presence of an activating agent; and wherein Zands T are as defined above; and wherein the term SUPPORT is defined asan organic or an inorganic substance.
 51. The process defined in claim50, wherein the moiety Q is unsubstituted.
 52. The process defined inclaim 50, wherein the moiety Q is substituted by at least one moietyselected from the group consisting of a C₁–C₄₀ alkyl group, a C₅–C₄₀aryl group, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀hydroxy-containing group, a C₂–C₄₀ acrylate-containing group and aC₅–C₄₀ alkylaryl group.
 53. The process defined in claim 50, wherein Thas the formula:

wherein q and s are the same or different and each is an integer havinga value of 0–40 and r is an integer having a value of 1–200.
 54. Theprocess defined in claim 53, wherein q and s are the same or differentand each is an integer having a value of 1–20 and r is an integer havinga value of 1–150.
 55. The process defined in claim 50, wherein T has theformula:

wherein a is 0 or 1, R^(a) is selected from —OH, —NH₂, —NHR and —ORwherein R is a protecting group and b is an integer having a value of0–40 and Q is a moiety having the formula:

wherein q r s, t and u are the same or different and each is an integerhaving a value of 0–40 and R^(a′) is selected from the group consistingof hydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, aC₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxy-containinggroup, a C₂–C₄₀ acrylate-containing group, a C₅–C₄₀ alkylaryl group,NH₂, —NHR and —OR, wherein R is a protecting group.
 56. The reusablelinker arm defined in claim 55, wherein a is 0 and R^(a) is —OH.
 57. Thereusable linker arm defined in claim 55, wherein a is 1 and R^(a) is —NRor —OR.
 58. The process defined in claim 55, wherein the protectinggroup is selected from the group consisting of acetyl, chloroacetyl,methoxyacetyl, t-butyl phenoxyacetyl, trityl, methoxytrityl,dimethoxytrityl (DMT), pivalyl-isobutyloxycarbonyl,t-butyldimethylsilyl, phenoxyacetal, 9-phenylxanthen-9-yl (pixyl),tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl,benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl,dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl,isopropyl-dimethylsilyl, diisopropylmethylsilyl, diethylisopropylsilyl,triisopropylsilyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl,o-nitrobenzyl, o-hydroxystyryldimethylsilyl, 2-oxo-1,2-diphenylethyl,allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl,fluorenyl-methoxycarbonyl, 2-phenylsulfonyl-ethoxycarbony,fluorophenyl-methoxypiperidinyl and mixtures thereof.
 59. The processdefined in claim 50, wherein p is
 0. 60. The process defined in claim50, wherein B¹ is selected from the group consisting of hydrogen,halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, a substitutedor unsubstituted C₅–C₃₀ aryl group and a substituted or unsubstitutedC₅–C₄₀ alkylaryl group.
 61. The process defined in claim 50, whereineach of R⁴, R⁵, R⁶ and R⁷ is hydrogen.
 62. The process defined in claim50, wherein each of m and n are
 1. 63. The process defined in claim 50,wherein each of R¹, R² and R³ is hydrogen.
 64. The process defined inclaim 50, wherein X¹ and X² are both —O—.
 65. The process defined inclaim 50, wherein SUPPORT is an inorganic substance.
 66. The processdefined in claim 65, wherein the inorganic substance is selected fromthe group consisting of silica, glass beads, porous glass,aluminosilicates, borosilicates, metal oxides, clays and mixturesthereof.
 67. The process defined in claim 50, wherein SUPPORT is anorganic substance.
 68. The process defined in claim 67, wherein theorganic substance is a cross-linked polymer.
 69. The process defined inclaim 50, wherein the activating agent is selected from the groupconsisting of an acid chloride; an active ester; an active hydroxylamineester; acid anhydride and mixed anhydride.
 70. The process defined inclaim 50, wherein the activating agent is selected from the groupconsisting of one or more arylsulfonyl chlorides; active arylsulfonylesters; 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ); one ormore acyl carbonates one or more 1,1′-(carbonyldioxybenzotriazoles;chlorotrimethylsilane one or more carbodiimides either alone or incombination with auxiliary nucleophiles and/or catalysts; or uroniumsalts a catalyst or phosphonium salts or mixtures thereof.
 71. A processfor production of a reusable linker arm for oligonucleotide synthesishaving the following formula:NUCLEOSIDE-Z-O-T

[SUPPORT] wherein Z is selected from the group consisting of:HO₂C—CH₂—CH₂—(C═O)—; HO₂C—CH₂—O—CH₂—(C═O)—; HO₂C—(C═O)—; and

wherein: R¹, R² and R³ are the same or different and are selected fromthe group consisting of hydrogen, halide, a substituted or unsubstitutedC₁–C₂₀ alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl groupand a substituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁴ and R⁵ arethe same or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; X¹ is selected from the groupconsisting of —O—, —S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected fromthe group consisting of hydrogen, a substituted or unsubstituted C₁–C₂₀alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; n is 0, 1 or 2; andone of A¹ and B¹ is selected from the group consisting of hydrogen,halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group and the other of A¹ and B ¹ has theformula:

wherein p is 0 or 1; X² is selected from the group consisting of —O—,—S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected from the groupconsisting of hydrogen, a substituted or unsubstituted C₁–C₂₀ alkylgroup, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁶ and R⁷ are thesame or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; and T has the formula:—[CH₂]_(q)—[O—CH₂—CH₂—O]_(r)—[CH₂]_(s)— wherein q and s are the same ordifferent and each is an integer having a value of 0–40 and r is aninteger having a value of 1–200 or T has the formula:-[Q]_(a)-CH₂—CH(R^(a))—CH₂—O—[CH₂]_(b)— wherein a is 0 or 1, R^(a) isselected from —OH, —NH₂, —NHR and —OR wherein R is a protecting groupand b is an integer having a value of 0–40, and Q is moiety having theformula:—[CH₂]_(u)—[CH(R^(a′))]_(t)—[CH₂]_(q)—O—[CH₂]_(r)—O—[CH₂]_(s)— whereinq, r, s, t and u are the same or different and each is an integer havinga value of 0–40 and R^(a′) is selected from the group consisting ofhydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, a C₁–C₄₀alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxy-containing group, aC₂–C₄₀ acrylate-containing group, a C₅–C₄₀ alkylaryl group, —NH₂, —NHRand —OR, wherein R is a protecting group, the process comprising asequence of steps or reacting together the compounds having Formulae I,II and III in the presence of an activating agentHO-Z-OH   (I)HO-T

[SUPPORT]  (II)NUCLEOSIDE-OH   (III) wherein Z ands T are as defined above and; whereinthe term SUPPORT is defined as an organic or an inorganic substance; andwherein the term NUCLEOSIDE represents an optionally protectedribonucleosidyl or 2′-deoxyribonucleosidyl group; with the proviso thatsaid compounds are reacted in the pairs I+II, or I+III, prior to thecoupling of the resultant intermediate product with the remainingcompound.
 72. The process defined in claim 71, wherein the moiety Q isunsubstituted.
 73. The process defined in claim 71, wherein the moiety Qis substituted by at least one moiety selected from the group consistingof a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, a C₁–C₄₀ alkoxy group, aC₁–C₄₀ ester group, a C₁–C₄₀ hydroxy group, a C₂–C₄₀ acrylate group anda C₅–C₄₀ alkylaryl group.
 74. The process defined in claim 71, wherein Thas the formula:

wherein q and s are the same or different and each is an integer havinga value of 0–40 and r is an integer having a value of 1–200.
 75. Theprocess defined in claim 74, wherein q and s are the same or differentand each is an integer having a value of 1–20 and r is an integer havinga value of 1–150.
 76. The process defined in claim 71, wherein T has theformula:

wherein a is 0 or 1, Q is an organic moiety, R^(a) is selected from —OH,—NH₂, —NHR and —OR wherein R is a protecting group and b is an integerhaving a value of 0–40, and Q is a moiety having the formula:

wherein q, r, s, t and u are the same or different and each is aninteger having a value of 0–40 and R^(a′) is selected from the groupconsisting of hydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ arylgroup, a C₁–C₄₀ alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀hydroxy-containing group, a C₂–C₄₀ acrylate-containing group, a C₅–C₄₀alkylaryl group, —NH₂, —NHR and —OR wherein R is a protecting group. 77.The reusable linker arm defined in claim 76, wherein a is 0 and R^(a) is—OH.
 78. The reusable linker arm defined in claim 76, wherein a is 1 andR^(a) is —NR or —OR.
 79. The process defined in claim 76, wherein theprotecting group is selected from the group consisting of acetyl,chloroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, trityl,methoxytrityl, dimethoxytrityl (DMT), pivalyl-isobutyloxycarbonyl,t-butyldimethylsilyl, phenoxyacetal, 9-phenylxanthen-9-yl (pixyl),tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl,benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl,dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl,isopropyl-dimethylsilyl, diisopropylmethylsilyl, diethylisopropylsilyl,triisopropylsilyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl,o-nitrobenzyl, o-hydroxystyryldimethylsilyl, 2-oxo-1,2-diphenylethyl,allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl,fluorenyl-methoxycarbonyl, 2-phenylsulfonyl-ethoxycarbony,fluorophenyl-methoxypiperidinyl and mixtures thereof.
 80. The processdefined in claim 71, wherein p is
 0. 81. The process defined in claim71, wherein B¹ is selected from the group consisting of hydrogen,halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, a substitutedor unsubstituted C₅–C₃₀ aryl group and a substituted or unsubstitutedC₅–C₄₀ alkylaryl group.
 82. The process defined in claim 71, whereineach of R⁴, R⁵R⁶ and R⁷ is hydrogen.
 83. The process defined in claim71, wherein each of m and n are
 1. 84. The process defined in claim 71,wherein each of R¹, R² and R³ is hydrogen.
 85. The process defined inclaim 71, wherein X¹ and X² are both —O—.
 86. The process defined inclaim 71, wherein SUPPORT is an inorganic substance.
 87. The processdefined in claim 86, wherein the inorganic substance is selected fromthe group consisting of silica, glass beads, porous glass,aluminosilicates, borosilicates, metal oxides, clays and mixturesthereof.
 88. The process defined in claim 71, wherein SUPPORT is anorganic substance.
 89. The process defined in claim 88, wherein theorganic substance is a cross-linked polymer.
 90. The process defined inclaim 71, wherein the activating agent comprises at least one memberselected from the group consisting of an acid chloride; an active ester;an active hydroxylamine ester; acid anhydride and mixed acid anhydride.91. The process defined in claim 71, wherein the activating agent isselected from the group consisting of one or more arylsulfonylchlorides, one or more active arylsulfonyl esters,2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ), one or moreacyl carbonates, one or more 1,1′-(carbonyldioxy)dibenzotriazoles,chlorotrimethylsilane, and one or more carbodiimides; alone or incombination with a catalyst, one or more uronium salts, or one or morephosphonium salts; or mixtures thereof, or mixtures thereof withauxiliary nucleophiles.
 92. The process defined in claim 71, whereinNUCLEOSIDE is a moiety selected from one of the following formulae:

wherein R⁸ and R¹⁰ are the same or different and are hydrogen or aprotecting group, R⁹ is hydrogen or —OR¹¹ wherein R¹¹ is hydrogen or aprotecting group, and B* is a nucleic acid base.
 93. The process definedin claim 71, wherein the compounds of Formulae I and II are initiallyreacted to form a conjugate which is reacted with the compound ofFormula III.
 94. The process defined in claim 71, wherein compounds ofFormulae I and III are initially reacted to form a conjugate which isreacted with the compound of Formula II.
 95. A process for producing anoligonucleotide having a desired sequence comprising the steps of: (i)reacting a linker arm having the formula:NUCLEOSIDE-Z-O-T

[SUPPORT] wherein Z is selected from the group consisting of:HO₂C—CH₂—CH₂—(C═O)—; HO₂C—CH₂—O—CH₂—(C═O)—; HO₂C—(C═O)—; and

wherein: R¹, R² and R³ are the same or different and are selected fromthe group consisting of hydrogen, halide, a substituted or unsubstitutedC₁–C₂₀ alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl groupand a substituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁴ and R⁵ arethe same or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₁–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; X¹ is selected from the groupconsisting of —O—, —S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected fromthe group consisting of hydrogen, a substituted or unsubstituted C₁–C₂₀alkyl group, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; n is 0, 1 or 2; andone of A¹ and B¹ is selected from the group consisting of hydrogen,halide, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group and the other of A¹ and B¹ has theformula:

wherein p is 0 or 1; X² is selected from the group consisting of —O—,—S—, —C(O)—, —S(O)₂—, and —N(R)—; R is selected from the groupconsisting of hydrogen, a substituted or unsubstituted C₁–C₂₀ alkylgroup, an substituted or unsubstituted C₅–C₃₀ aryl group and asubstituted or unsubstituted C₅–C₄₀ alkylaryl group; R⁶ and R⁷ are thesame or different and are selected from the group consisting ofhydrogen, a substituted or unsubstituted C₁–C₂₀ alkyl group, ansubstituted or unsubstituted C₅–C₃₀ aryl group and a substituted orunsubstituted C₅–C₄₀ alkylaryl group; and T has the formula:—[CH₂]_(q)—[O—CH₂—CH₂—O]_(r)[CH₂]_(s)— wherein q and s are the same ordifferent and each is an integer having a value of 0–40 and r is aninteger having a value of 1–200 or T has the formula:-[Q]_(a)-CH₂—CH(R^(a))—CH₂—O—[CH₂]_(b)— wherein a is 0 or 1, R^(a) isselected from —OH, —NH₂, —NHR and —OR wherein R is a protecting groupand b is an integer having a value of 0–40, and Q is moiety having theformula:—[CH₂]_(u)—[CH(R^(a′))]_(t)—[CH₂]_(q)—O—[CH₂]_(r)—O—[CH₂]_(s)— whereinq, r, s, t and u are the same or different and each is an integer havinga value of 0–40 and R^(a′) is selected from the group consisting ofhydrogen, hydroxyl, a C₁–C₄₀ alkyl group, a C₅–C₄₀ aryl group, a C₁–C₄₀alkoxy group, a C₁–C₄₀ ester group, a C₁–C₄₀ hydroxy-containing group, aC₂–C₄₀ acrylate-containing group, a C₅–C₄₀ alkylaryl group, —NH₂, —NHRand —OR, wherein R is a protecting group, with at least one activatednucleotide monomer until an oligonucleotide having the desired sequenceis produced; (ii) cleaving the oligonucleotide having the desiredsequence to produce a free oligonucleotide having the desired sequence;and a used linker arm; and (iii) isolating the used linker arm; andwherein the term SUPPORT is defined as an organic or an inorganicsubstance; and wherein the term NUCLEOSIDE represents an optionallyprotected ribonucleosidyl or 2′-deoxyribonucleosidyl group.
 96. Theprocess defined in claim 95, wherein the used linker arm produced inStep (ii) has the formula:Z-O-T

[SUPPORT] wherein Z, T and SUPPORT are as defined in claim
 95. 97. Theprocess defined in claim 95, wherein Step (iii) further comprises theadditional step of converting the used linked arm to a linker arm havingthe formula:NUCLEOSIDE-Z-O-T

[SUPPORT] wherein Z, T, NUCLEOSIDE and SUPPORT are as defined in claim95; and wherein the additional step comprises contacting the used linkerarm with an activating agent in the presence of an appropriatelyprotected NUCLEOSIDE.